Biological research today is increasingly focusing on determining the positions within the cell of various cellular components to ever higher spatial resolutions. This involves many different techniques for enhancing resolution and contrast in images, both for electron microscopes (TEMs, STEMs, SEMs, etc.), as well as all types of light microscopes, including the latest super-resolution techniques. One powerful technique that has gained wide acceptance for research into cellular structure, transport, metabolism, and motility is the application of recombinant genetic techniques to link “reporter” genes to “genes of interest” (GoIs). Thus, when a particular GoI is expressed during normal genetic transcription/translation processes, the reporter gene will also be expressed, producing a small protein which ends up attached to the “protein of interest” (PoI) encoded for by the GoI. One widely-accepted reporter gene is that encoding for a green fluorescent protein (GFP), these reporter genes being available in wild and genetically-modified versions, and the expressed GFPs having fluorescence that extends from blue to yellow in emission wavelengths. The GFP is relatively small (29.6 kDa, 3 nm in diameter by 4 nm long) with its chromophore well protected inside and not requiring any co-factors for light emission. All that is needed to “light up” a GFP is illumination by a laser with a slightly shorter wavelength than the GFP emission wavelength. GFPs appear to be essentially “inert” to the proper functioning of their attached PoI—this is ensured in some cases by connecting the GFP to the PoI with a short flexible polypeptide “linker” which enables the GFP to swing around free from the protein, which may be part of some intracellular structure or mechanism that must not be interfered with in order to preserve the cellular functions being studied by the researcher.
Clearly, if the X-Y-Z location of the GFP can be determined precisely within a cell (say, to 10 nm accuracy) then the location of the PoI would be known to a similar accuracy. The fluorescing GFP can be observed through a light microscope and so the location of the PoI can be seen in the microscope relative to observable structures in the sample. Several techniques in the prior art have been proposed and, in some cases, demonstrated, for achieving high positional information from various fluorescent markers (FMs) such as GFPs and also quantum dots. In one technique, a green laser is used to excite a small portion of the fluorescent markers (FMs) in a sample, and the sample is then imaged. Using Gaussian curve fitting, the locations of the FMs may be determined within a FWHM of 20 nm, substantially smaller than the diffraction limit of the imaging system. Using multiple green laser flashes, alternating with red laser flashes which extinguish the fluorescence, the locations of a larger number of FMs may be determined in a process which may typically take tens of minutes. In another technique, described in U.S. Pat. No. 7,317,515 to Buijsse et al. for “Method of Localizing Fluorescent Markers,” which is assigned to the assignee of the present application and which is hereby incorporated by reference, a charged particle beam scans the surface of the sample, damaging the markers and extinguishing the fluorescence when the beam hits the FM. The location of the FM corresponds to the position of the charged particle beam when the fluorescence is extinguished. Because the charged particle beam can be focused to a much smaller point than the laser that illuminates the marker, and the position of the charged particle beam at any time during its scan can be determined with great accuracy, the position of the FM, and therefore the position of the PoI, can be determined with similar accuracy.
Throughout all descriptions herein of the present invention, the term GFP will be used to represent the larger class of FMs which can be damaged by a charged particle beam (comprising electrons or ions), including GFPs, organic dyes, as well as inorganic markers such as quantum dots (which may typically be functionalized to enable selective attachment to particular intracellular components such as proteins, nucleic acids, etc.).
Many of the prior art methods for localization of FMs within biological samples work only for relatively small numbers of FMs, from which a small subset are activated at any one time—thus imaging times can be many minutes and still suffer from some of the limitations of light optical imaging. Prior art methods employing charged particle beams to selectively damage FMs within samples have utilized image processing methods capable of dealing only with relatively small numbers of FMs—for these methods, the statistical signal-to-noise ratio limits their application to FMs which do not inherently appear in large densities. For GFPs, in particular, this may be a hindrance, since any type of expressible tag (as opposed to a functionalized tag such as a quantum dot) can be created in very large numbers through normal cellular process of gene transcription to mRNA, followed by translation to proteins (GoI+linker+GFP). Thus, there is a need for a fast method for localization of very large numbers (≧10000) of FMs such as GFPs within cells, or sections of cells, in time frames, for example, of the order of a minute.